EP3781673A1 - Méthode permettant d'améliorer les propriétés suppressives de lymphocytes treg - Google Patents

Méthode permettant d'améliorer les propriétés suppressives de lymphocytes treg

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Publication number
EP3781673A1
EP3781673A1 EP19719600.9A EP19719600A EP3781673A1 EP 3781673 A1 EP3781673 A1 EP 3781673A1 EP 19719600 A EP19719600 A EP 19719600A EP 3781673 A1 EP3781673 A1 EP 3781673A1
Authority
EP
European Patent Office
Prior art keywords
treg
foxp3
cells
tregs
polynucleotide encoding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19719600.9A
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German (de)
English (en)
Inventor
Hans Stauss
Jenny L. MCGOVERN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
UCL Business Ltd
Original Assignee
UCL Business Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB1806331.3A external-priority patent/GB201806331D0/en
Priority claimed from GBGB1806330.5A external-priority patent/GB201806330D0/en
Application filed by UCL Business Ltd filed Critical UCL Business Ltd
Publication of EP3781673A1 publication Critical patent/EP3781673A1/fr
Pending legal-status Critical Current

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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
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Definitions

  • the present invention relates to a method for enhancing the ability of regulatory T cells (Tregs) to suppress immune responses.
  • the present invention relates to a method of increasing FOXP3 expression in Tregs.
  • the present invention further relates to an engineered Treg provided by the method of the invention and to methods and uses of such an engineered Treg.
  • Tregs are a type of T cell that modulates the activity of the immune system.
  • Tregs are immunosuppressive, down-regulating immune responses to stimuli.
  • Tregs suppress induction and proliferation of conventional T cells, some types of which are directly involved in immune responses (e.g. cytotoxic T cells).
  • the suppressive effect of Tregs can be directed towards specific antigens by expression of recombinant T cell receptor (TOR) constructs that recognise peptides matching epitopes found within the antigen in question.
  • TOR recombinant T cell receptor
  • the suppressive effect of Tregs can be directed towards specific targets by expression of chimeric antigen receptors (CARs) that recognise antigens expressed on the surface of target cells.
  • CARs chimeric antigen receptors
  • Conventional T cells can be differentiated towards a regulatory phenotype ex-vivo by expressing FOXP3 in said cells.
  • autoimmune and inflammatory central nervous system (CNS) diseases the immune system attacks self-antigens.
  • CNS central nervous system
  • MS Multiple Sclerosis
  • the immune system attacks the myelin sheath of neurons of the central nervous system.
  • autoimmune and inflammatory CNS diseases generally suppress the immune system.
  • one treatment includes transplantation of bone marrow along with administration of cytostatics and immunosupressive drugs.
  • Autologous haematopoietic stem cell transplantation can have lasting beneficial effects for some subjects, but the procedure requires aggressive myelo-ablative conditioning which is associated with substantial toxicity and risk.
  • DMTs disease-modifying treatments
  • stem cell transplantation can mediate CNS-specific suppression of the immunopathology of autoimmune and inflammatory CNS diseases.
  • effective treatments for autoimmune and inflammatory CNS diseases do not exist. Treatment is focused on merely reducing its symptoms, usually by general suppression of the immune system. There is a need for a therapy which specifically targets local immune responses associated with onset and progression of CNS disease.
  • the present inventors have surprisingly found that exogenous FOXP3 expression in regulatory T cell (Tregs) (which already express endogenous FOXP3) enhances their regulatory function.
  • the invention provides a method for enhancing the ability of regulatory T cells (Tregs) to suppress immune responses.
  • the Tregs of the present invention are natural Tregs or induced Tregs which developed from conventional T cells.
  • the Tregs of the present invention are natural Tregs or induced Tregs which developed from conventional T cells in vivo.
  • Suitable Treg cells include thymus-derived, natural Treg (nTreg) cells and peripherally generated, induced Treg (iTreg) cells.
  • the Tregs of the present invention express endogenous FOXP3.
  • the present inventors have determined that increasing FOXP3 expression in Tregs which already express endogenous FOXP3 (e.g. by introducing exogenous FOXP3) enhances the regulatory function of the Tregs to a greater degree than the regulatory function provided by expressing exogenous FOXP3 in conventional T cells which do not express endogenous FOXP3.
  • the present inventors have further determined that increasing FOXP3 expression in Tregs which already express endogenous FOXP3 enables improved retention of a Treg functional profile in vivo following administration to a subject.
  • natural Tregs which do not express exogenous FOXP3 may lose their Treg profile following administration to a subject - for example natural Tregs which do not express exogenous FOXP may have reduced levels of FOXP3 expression and be capable of producing pro- inflammatory, effector cytokines after a period following administration to a subject.
  • Tregs provided by the present invention may retain FOXP3 expression and have reduced capability to produce pro-inflammatory, effector cytokines after a period following administration to a subject.
  • the Tregs of the present invention are natural Tregs.
  • the invention provides a method for generating a population of regulatory T cells (Tregs) comprising providing a first population of Tregs and increasing FOXP3 expression in the first population of Tregs to generate a second population of Tregs.
  • Tregs regulatory T cells
  • the invention provides a method for enhancing the ability of a Tregs to suppress immune responses comprising increasing FOXP3 expression in the Treg.
  • FOXP3 expression is increased by introducing into the Tregs a polynucleotide encoding a FOXP3 protein.
  • the method for enhancing the ability of regulatory T cells (Tregs) to suppress immune responses comprises:
  • the Treg may refer to a population of Tregs (i.e. a plurality of Tregs).
  • the invention also provides an engineered Treg obtainable or obtained by the method of the invention.
  • the invention also provides an engineered Treg having higher FOXP3 expression than a non-engineered Treg.
  • the engineered Treg comprises an exogenous polynucleotide encoding a FOXP3 protein.
  • the invention also provides a pharmaceutical composition comprising an engineered Treg of the invention.
  • the invention also provides an engineered Treg of the invention or a pharmaceutical composition of the invention for use in prevention and/or treatment of a disease.
  • the invention also provides the use of an engineered Treg of the invention in the manufacture of a medicament.
  • the invention also provides a method for prevention and/or treatment of a disease comprising administering to a subject an engineered Treg or a composition of the invention.
  • Figure 1 shows the proliferation of Tconv cells transduced with a TOR construct, with and without peptide (shown with“*”), and the proliferation of the same cells in the presence of mock-transduced Tregs (white bars), Tregs transduced with a TCR construct (black bars) or Tregs transduced with a TCR construct and FOXP3 (grey bars) at different Treg:Tconv ratios.
  • Figure 2 shows the IL-2 production of Tconv cells transduced with a TCR construct, with and without peptide (shown with “*”), and the IL-2 production of the same cells in the presence of mock-transduced Tregs (white bars), Tregs transduced with a TCR construct (black bars) or Tregs transduced with a TCR construct and FOXP3 (grey bars) at different Treg:Tconv ratios.
  • Figure 3 shows the proliferation of Tconv cells (from a different donor to Figure 1) transduced with a TCR construct, with and without peptide (shown with “*”), and the proliferation of the same cells in the presence of mock-transduced Tregs (white bars), Tregs transduced with a TCR construct (black bars) or Tregs transduced with a TCR construct and FOXP3 (grey bars) at different Treg:Tconv ratios.
  • Figure 4 shows the IL-2 production of Tconv cells (from a different donor to Figure 2) transduced with a TCR construct with and without peptide (shown with “*”) and the proliferation of the same cells in the presence of mock-transduced Tregs (white bars), Tregs transduced with a TCR construct (black bars) or Tregs transduced with a TCR construct and FOXP3 (grey bars) at different Treg:Tconv ratios.
  • Figure 5 shows the mean fluorescence intensity (MFI) of Treg markers (FOXP3, CD25 and CTLA-4) in mock-transduced Treg or Treg transduced with TCR or TCR+FOXP3 analysed by flow cytometry at d7-10 after transduction. Dots are representative of individual experiments. 1-way ANOVA was used for statistical analysis p ⁇ 0.05 *, p ⁇ 0.005 **.
  • Figure 6 shows the MFI of FOXP3, CD25 and CTLA-4 in transduced Tregs. Each line represents a single experiment showing MFI of markers on the same Treg transduced with TCR or TCR+FOXP3.
  • Figure 7 shows the proliferation of Tconv cells (from a different donor to Figures 1 and 3) transduced with a TCR construct, with and without peptide, and the proliferation of the same cells in the presence of mock-transduced Tregs (white bars), Tregs transduced with a TCR construct (black bars), Tregs transduced with a TCR construct and FOXP3 (grey bars) or Tconv cells transduced with a TCR construct and FOXP3, i.e. induced Tregs (red bars, right- hand bar of each data set), at different Treg:Tconv ratios.
  • Figure 8 shows the level of IL-2 production by Tconv cells (from the same donor as Figure 7) transduced with a TCR construct, with and without peptide, and IL-2 production by the same cells in the presence of mock-transduced Tregs (white bars), Tregs transduced with a TCR construct (black bars), Tregs transduced with a TCR construct and FOXP3 (grey bars) or Tconv transduced with a TCR construct and FOXP3, i.e. induced Tregs (red bars right- hand bar of each data set), at different Treg:Tconv ratios.
  • FIG. 9 TCR transduced regulatory T cells can engraft into irradiated hosts but require exogenous FOXP3 expression to prevent accumulation of TCR+FOXP3- cells.
  • Thy1.1+CD4+CD25+ Treg were isolated from lymph nodes and splenocytes of HLA- DRB*0401 transgenic mice by bead sort. Treg were transduced TCR, TCR+murine FOXP3 or cultured with virus-free supernatant (mock). 1 day after transduction TCR or TCR+FOXP3 transduced cells were injected into HLA-DRB*0401 transgenic hosts conditioned with 4Gy irradiation. 7 weeks later flow cytometry was used to determine the engraftment of transduced Treg A. Transduction efficiency was determined through expression of human variable 2.1 and murine Foxp3 on d1 post-transduction B.
  • Splenocytes from mice that received Treg transduced with TCR or TCR+FOXP3 were stained with Thy1.1 to identify transferred cells (top panel) and FOXP3 and TCR (bottom panel)
  • Statistical analysis by unpaired t test D Representative expression of FOXP3 within transduced cells 7 weeks after transfer.
  • FIG 10 - Treg expressing exogenous FOXP3 retain Treg functionality after 7 weeks in vivo whilst Tregs not expressing exogenous FOXP3 acquire the ability to produce effector cytokines
  • a Splenocytes were cultured for 4 hours with CD86+HLA-DR4+CHO cells pulsed with irrelevant peptide or 10uM MBP. Production of IL-2 and IFNg was determined by flow cytometry. FACS plots show CD45.1 cells (top panel) containing Treg expressing TCR alone and Thy1.1 cells containing Treg expressing TCR+FOXP3. B Graphs show cumulative IL-2 and IFNg production by TCR-expressing (dark grey) and TCR+FOXP3-expressing (light grey) Treg.
  • Figure 11 - shows a schematic diagram of an illustrative retroviral vector encoding (A) TCR alpha and beta chains and (B) FOXP3 plus TCR alpha and beta chains.
  • the invention provides a method for enhancing the ability of a regulatory T cell (Tregs) to suppress immune responses comprising increasing FOXP3 expression the Treg.
  • Tregs regulatory T cell
  • Treg means a T cell which expresses the markers CD4, CD25 and FOXP3 (CD4 + CD25 + FOXP3 + ).
  • Tregs may be identified using the cell surface markers CD4 and CD25 in the absence of or in combination with low-level expression of the surface protein CD127 (CD4 + CD25 + CD127 or CD4 + CD25 + CD127
  • Tregs may also express on the cell surface high levels of CTLA-4 (cytotoxic T-lymphocyte associated molecule-4) or GITR (glucocorticoid-induced TNF receptor). Unlike conventional T cells, Tregs do not produce IL-2 and are therefore anergic at baseline.
  • CTLA-4 cytotoxic T-lymphocyte associated molecule-4
  • GITR glucocorticoid-induced TNF receptor
  • Natural Treg means a thymus-derived Treg. Natural Tregs are CD4 + CD25 + FOXP3 + Helios + Neuropilin1 + .
  • the term “natural Treg” distinguishes thymus- derived Tregs from“induced Tregs”, which develop from conventional T cells outside the thymus. Compared with induced Tregs, natural Tregs have higher expression of PD-1 (programmed cell death-1 , pdcdl), neuropilin 1 (Nrp1), Helios (Ikzf2), and CD73. Natural Tregs may be distinguished from induced Tregs on the basis of the expression of Helios protein or Neuropilin 1 (Nrp1) individually.
  • iTreg induced regulatory T cell
  • iTreg means a CD4 + CD25 + FOXP3 + Helios Neuropilin T T cell which develops from mature CD4+ conventional T cells outside of the thymus.
  • iTregs can be induced in vitro from CD4+ CD25-FOXP3- cells in the presence of IL-2 and TGF-b.
  • the Treg expresses FOXP3 from the endogenous FoxP3 gene of the cell.
  • the Treg may be a CD4 + CD25 + FOXP3 + Treg.
  • the Treg may be a CD4 + CD25 + CD127 Treg.
  • the Treg may be a CD4 + CD25 + CD127
  • the Treg may be a CD4 + CD25 + CD127 CD45RA + Treg.
  • the Treg may be a CD4 + CD25 + CD127
  • the Treg may be a CD4 + CD25 + FOXP3 + CD127 Treg.
  • the Treg may be a CD4 + CD25 + FOXP3 + CD127 low Treg.
  • the Treg is a CD4 + CD25 + FOXP3 + Helios + Treg.
  • the Treg is a CD4 + CD25 + FOXP3 + Neuropilin 1 + Treg.
  • the Treg is a CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + Treg.
  • the Treg is a human Treg.
  • the Treg is a human Treg and the FOXP3 is human FOXP3.
  • Tconv cells meaning conventional T cells, refers to T cells that are not Tregs.
  • the Treg of the present invention may be derived from a stem cell.
  • the Treg of the present invention may be derived from a stem cell in vitro.
  • the cell is a progenitor cell.
  • stem cell means an undifferentiated cell which is capable of indefinitely giving rise to more stem cells of the same type, and from which other, specialised cells may arise by differentiation.
  • Stem cells are multipotent. Stem cells may be for example, embryonic stem cells or adult stem cells.
  • progenitor cell means a cell which is able to differentiate to form one or more types of cells but has limited self-renewal in vitro.
  • the cell is capable of being differentiated into a T cell, such as a Treg.
  • the cell has the ability to differentiate into a T cell, which expresses FOXP3 such as a Treg.
  • the cell may be an embryonic stem cell (ESC).
  • the cell is a haematopoietic stem cell or haematopoietic progenitor cell.
  • the cell is an induced pluripotent stem cell (iPSC).
  • iPSC induced pluripotent stem cell
  • the cell may be obtained from umbilical cord blood.
  • the cell may be obtained from adult peripheral blood.
  • hematopoietic stem and progenitor cell (HSPCs) may be obtained from umbilical cord blood.
  • Cord blood can be harvested according to techniques known in the art (e.g., U.S. Pat. Nos. 7,147,626 and 7,131 ,958 which are incorporated herein by reference).
  • HSPCs may be obtained from pluripotent stem cell sources, e.g., induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).
  • pluripotent stem cell sources e.g., induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).
  • iPSCs induced pluripotent stem cells
  • ESCs embryonic stem cells
  • the term“hematopoietic stem and progenitor cell” or“HSPC” refers to a cell which expresses the antigenic marker CD34 (CD34+) and populations of such cells.
  • the term“HSPC” refers to a cell identified by the presence of the antigenic marker CD34 (CD34+) and the absence of lineage (lin) markers.
  • the population of cells comprising CD34+ and/or Lin(-) cells includes haematopoietic stem cells and hematopoietic progenitor cells.
  • HSPCs can be obtained or isolated from bone marrow of adults, which includes femurs, hip, ribs, sternum, and other bones. Bone marrow aspirates containing HSPCs can be obtained or isolated directly from the hip using a needle and syringe. Other sources of HSPCs include umbilical cord blood, placental blood, mobilized peripheral blood, Wharton's jelly, placenta, fetal blood, fetal liver, or fetal spleen. In particular embodiments, harvesting a sufficient quantity of HSPCs for use in therapeutic applications may require mobilizing the stem and progenitor cells in the subject.
  • iPSC induced pluripotent stem cell
  • HSC and HPC hematopoietic stem or progenitor cell
  • reprogramming refers to a method of increasing the potency of a cell to a less differentiated state.
  • the term“programming” refers to a method of decreasing the potency of a cell or differentiating the cell to a more differentiated state.
  • the expression“enhancing the ability to suppress immune responses” means to increase the suppressive effect of a Treg (or population of such Tregs) on an immune response in comparison to the suppressive effect of a corresponding Treg which has not been modified by a method of the invention (or population of such Tregs).
  • the term “immune response” refers to a number of physiological and cellular effects facilitated by the immune system in response to a stimulus such as a pathogen or an autoantigen. Examples of such effects include increased proliferation of Tconv cells and secretion of cytokines. Any such effects may be used as indicators of the strength of an immune response.
  • a relatively weaker immune response by Tconv in the presence of modified Tregs compared to non-modified Treg would indicate a relative enhancement of the modified Tregs to suppress immune responses.
  • a relative decrease in cytokine secretion would be indicative of a weaker immune response, and thus an enhancement of the ability of Tregs to suppress immune responses.
  • Assays are known in the art for measuring indicators of immune response strength, and thereby the suppressive ability of Tregs.
  • antigen-specific Tconv cells may be co-cultured with Tregs, and a peptide of the corresponding antigen added to the co-culture to stimulate a response from the Tconv cells.
  • the degree of proliferation of the Tconv cells and/or the quantity of the cytokine IL-2 they secrete in response to addition of the peptide may be used as indicators of the suppressive abilities of the co-cultured Tregs.
  • Antigen-specific Tconv cells co-cultured with Tregs of the present invention having increased FOXP3 expression may proliferate 5, 10, 15, 20, 25, 30, 35 or 40 % less than the same Tconv cells co-cultured with corresponding Tregs that do not have increased FOXP3 expression.
  • Antigen-specific Tconv cells co-cultured with Tregs of the invention having increased FOXP3 expression may show a reduction of effector cytokine that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% greater than corresponding Tconv cells co-cultured with corresponding Tregs that do not have increased FOXP3 expression.
  • Antigen-specific Tconv cells co-cultured with Tregs of the invention having increased FOXP3 expression may produce 10%, 20%, 30%, 40%, 50%, 60% or less effector cytokine than than corresponding Tconv cells co-cultured with corresponding Tregs that do not have increased FOXP3 expression.
  • the effector cytokine may be selected from IL-2, IL-17, TNFa, GM-CSF,, IFN-g, IL-4, IL-5, IL-9, IL-10 and IL-13.
  • the effector cytokine may be selected from IL-2, IL-17, TNFa, GM-CSF and IFN-y.
  • Antigen-specific Tconv cells co-cultured with Tregs of the invention having increased FOXP3 expression may achieve suppression of IL-2 production at 1 ⁇ 2, 1/4, 1/8, 1/10 or 1/20 the cell number of corresponding Tregs that do not have increased FOXP3 expression.
  • FOXP3 is the abbreviated name of the forkhead box P3 protein.
  • FOXP3 is a member of the FOX protein family of transcription factors and functions as a master regulator of the regulatory pathway in the development and function of regulatory T cells.
  • Increasing FOXP3 expression means to increase the levels of FOXP3 mRNA and/or protein in a Treg (or population of such Tregs) in comparison to a corresponding Treg which has not been modified by a method of the invention (or population of such Tregs).
  • the level of FOXP3 mRNA and/or protein in a Treg modified by a method of the invention (or a population of such Tregs) may be increased to at least 1.5-fold, at least 2- fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 150-fold greater than the level in a corresponding Treg which has not been modified by a method of the invention (or population of such Tregs).
  • the level of FOXP3 mRNA and/or protein in a Treg modified by a method of the invention may be increased to at least 1.5-fold greater than the level in a corresponding Treg which has not been modified by a method of the invention (or population of such Tregs).
  • the level of FOXP3 mRNA and/or protein in a Treg modified by a method of the invention may be increased to at least 2-fold greater than the level in a corresponding Treg which has not been modified by a method of the invention (or population of such Tregs).
  • the level of FOXP3 mRNA and/or protein in a Treg modified by a method of the invention may be increased to at least 5-fold greater than the level in a corresponding Treg which has not been modified by a method of the invention (or population of such Tregs).
  • mRNA levels in a population of cells may be measured by techniques such as the Affymetrix ebioscience prime flow RNA assay, Northern blotting, serial analysis of gene expression (SAGE) or quantitative polymerase chain reaction (qPCR).
  • Protein levels in a population of cells may be measured by techniques such as flow cytometry, high- performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), Western blotting or enzyme-linked immunosorbent assay (ELISA).
  • FOXP3 expression is increased by introducing into the isolated Tregs a polynucleotide encoding a FOXP3 polypeptide.
  • introduction refers to methods for inserting foreign DNA into a cell, including both transfection and transduction methods.
  • Transfection is the process of introducing nucleic acids into a cell by non-viral methods.
  • Transduction is the process of introducing foreign DNA into a cell via a viral vector.
  • A“FOXP3 polypeptide” is a polypeptide having FOXP3 activity i.e. a polypeptide able to bind FOXP3 target DNA and function as a transcription factor regulating development and function of Tregs.
  • Techniques for measuring transcription factor activity are well known in the art.
  • transcription factor DNA-binding activity may be measured by ChIP.
  • the transcription regulatory activity of a transcription factor may be measured by quantifying the level of expression of genes which it regulates. Gene expression may be quantified by measuring the levels of mRNA and/or protein produced from the gene using techniques such as Northern blotting, SAGE, qPCR, HPLC, LC/MS, Western blotting or ELISA.
  • Genes regulated by FOXP3 include cytokines such as IL-2, IL-4 and IFN-g (Siegler et al. Annu. Rev. Immunol. 2006, 24: 209-26, incorporated herein by reference).
  • polynucleotide and“nucleic acid” are intended to be synonymous with each other.
  • a polynucleotide may be any suitable type of nucleotide sequence, such as a synthetic RNA/DNA sequence, a cDNA sequence or a partial genomic DNA sequence.
  • polypeptide is synonymous with "protein” and means a series of residues, typically L-amino acids, connected to one another typically by peptide bonds between the a- amino and carboxyl groups of adjacent amino acids.
  • polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code.
  • the skilled person may make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.
  • the polynucleotide may comprise DNA or RNA, may be single-stranded or double-stranded and may include synthetic or modified nucleotides.
  • a number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • Polynucleotides may be modified by any method in the art. Such modifications may enhance the in vivo activity or life span of the polynucleotide.
  • the polynucleotide may be in isolated or recombinant form. It may be incorporated into a vector and the vector may be incorporated into a host cell.
  • the polynucleotide may be codon optimised. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression.
  • the polynucleotide may be codon optimised for expression in a murine model of disease.
  • the polynucleotide may be codon optimised for expression in a human subject.
  • Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms. Codon optimisation may also involve the removal of mRNA instability motifs and cryptic splice sites.
  • the FOXP3 polypeptide may comprise the polypeptide sequence of a human FOXP3, such as UniProtKB accession Q9BZS1 , or a functional fragment thereof:
  • the FOXP3 polypeptide comprises an amino acid sequence which is at least 80 % identical to SEQ ID NO: 3 or a functional fragment thereof.
  • the FOXP3 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO: 3 or a functional fragment thereof.
  • the FOXP3 polypeptide comprises SEQ ID NO: 3 or a functional fragment thereof.
  • the FOXP3 polypeptide may be a variant of SEQ ID NO: 3, for example a natural variant.
  • the FOXP3 polypeptide is an isoform of SEQ ID NO: 3.
  • the FOXP3 polypeptide may comprise a deletion of amino acid positions 72-106 relative to SEQ ID NO: 3.
  • the FOXP3 polypeptide may comprise a deletion of amino acid positions 246-272 relative to SEQ ID NO: 3.
  • the F0XP3 polypeptide comprises SEQ ID NO: 4 or a functional fragment thereof:
  • TPGPEGRGSLLTCGDVEEN (SEQ ID NO: 4).
  • the FOXP3 polypeptide comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 4 or a functional fragment thereof.
  • the polypeptide comprises an amino acid sequence which is 85, 90, 95, 98 or 99% identical to SEQ ID NO: 4 or a functional fragment thereof.
  • the FOXP3 polypeptide may be a variant of SEQ ID NO: 4, for example a natural variant.
  • the FOXP3 polypeptide is an isoform of SEQ ID NO: 4 or a functional fragment thereof.
  • the FOXP3 polypeptide may comprise a deletion of amino acid positions 72-106 relative to SEQ ID NO: 4.
  • the FOXP3 polypeptide may comprise a deletion of amino acid positions 246-272 relative to SEQ ID NO: 4.
  • the FOXP3 polypeptide is encoded by the polynucleotide sequence set forth in SEQ ID NO: 1 :
  • the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence which is at least 80 % identical to SEQ ID NO: 1 or a functional fragment thereof.
  • the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO: 1 or a functional fragment thereof.
  • the polynucleotide encoding the FOXP3 polypeptide or variant comprises SEQ ID NO: 1 or a functional fragment thereof.
  • the F0XP3 polypeptide is encoded by the polynucleotide sequence set forth in SEQ ID NO: 2:
  • CTGCTAACATGCGGTGACGTCGAGGAGAATCC (SEQ ID NO: 2).
  • the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence which is at least 80 % identical to SEQ ID NO: 2 or a functional fragment thereof.
  • the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO: 2 or a functional fragment thereof.
  • the polynucleotide encoding the FOXP3 polypeptide or variant comprises SEQ ID NO: 2 or a functional fragment thereof.
  • the polynucleotide encoding the FOXP3 polypeptide or variant thereof may be codon optimised.
  • the polynucleotide encoding the FOXP3 polypeptide or variant thereof may be codon optimised for expression in a human cell.
  • Sequence comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate sequence identity between two or more sequences.
  • Sequence identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an“ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
  • sequence identity may be determined across the entirety of the sequence.
  • sequence identity may be determined across the entirety of the candidate sequence being compared to a sequence recited herein.
  • the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs).
  • GCG Wsconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details).
  • the public default values for the GCG package, or in the case of other software the default matrix, such as BLOSUM62 are used.
  • the polynucleotide encoding FOXP3 is a contiguous portion of an expression vector.
  • expression vector means a construct enabling expression of the FOXP3 polypeptide.
  • the expression vector is a cloning vector.
  • Suitable vectors may include, but are not limited to, plasmids, viral vectors, transposons, or nucleic acid complexed with polypeptide or immobilised onto a solid phase particle.
  • the expression vector is capable of sustained high-level expression in host cells.
  • the expression vector may be a retroviral vector.
  • the expression vector may be based on or derivable from the MP71 vector backbone.
  • the expression vector may lack a full-length or truncated version of the Woodchuck Hepatitis Response Element (WPRE).
  • WPRE Woodchuck Hepatitis Response Element
  • the vector also encodes a T cell receptor (TCR).
  • TCR T cell receptor
  • a TCR is a cell surface molecule that binds fragments of antigen bound to major histocompatibility complex (MHC) molecules on antigen presenting cells as part of directing an immune response.
  • MHC major histocompatibility complex
  • the TCR may be a recombinant protein, in other words the TCR may be an exogenous protein which is not naturally expressed by the present Treg of the invention.
  • the vector also encodes a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • a CAR is a recombinant cell surface molecule expressed by an engineered T cell that binds antigen expressed on the surface of other cells as part of directing an immune response. More particularly, CARs are proteins which graft the specificity of an antigen binder, such as a monoclonal antibody (mAb), to the effector function of a T-cell.
  • mAb monoclonal antibody
  • Their usual form is that of a type I transmembrane domain protein with an antigen recognizing amino terminus, a spacer, a transmembrane domain all connected to a compound endodomain which transmits T-cell survival and activation signals.
  • the vector comprises a polynucleotide encoding a TCR or CAR in addition to a polynucleotide encoding FOXP3; the vector may have the orientation of: 5’ FOXP3 - TCR/CAR 3’. Accordingly the polynucleotide encoding a FOXP3 may be 5’ to the polynucleotide encoding CAR or TCR.
  • the polynucleotide encoding FOXP3 may be separated from the polynucleotide encoding a TCR or CAR by a nucleic acid sequence which enables both the nucleic acid sequence encoding FOXP3 and the nucleic acid sequence encoding the TCR or CAR to be expressed from the same mRNA transcript.
  • the polynucleotide may comprise an internal ribosome entry site (IRES) between the nucleic acid sequences which encode (i) FOXP3 and (ii) the TCR or CAR.
  • IRES is a nucleotide sequence that allows for translation initiation in the middle of a mRNA sequence.
  • the polynucleotide may comprise a nucleic acid sequence encoding (i) FOXP3 and (ii) the TCR or CAR linked by an internal self-cleaving sequence.
  • the vector may have the structure: 5’ Strong promoter (e.g. LTR)-FoxP3-2A- CAR/TCR-3’LTR.
  • LTR strong promoter
  • FOXP3 expression is directly driven by the strong LTR promoter for optimal expression.
  • CAR/TCR is preceded by a 2A sequence and expression of the CAR/TCR is thus dependent on both LTR promoter activity and 2A cleavage activity.
  • a configuration in which FOXP3 precedes CAR/TCR in the 5’ to 3’ direction ensures that CAR/TCR expression can only occur when FOXP3 has been expressed and that expression of CAR/TCR without FOXP3 does not occur.
  • the internal self-cleaving sequence may be any sequence which enables the polypeptide comprising (i) FOXP3 and (ii) the TCR or CAR to become separated.
  • the cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual peptides without the need for any external cleavage activity.
  • cleavage is used herein for convenience, but the cleavage site may cause the peptides to separate into individual entities by a mechanism other than classical cleavage.
  • FMDV Foot-and-Mouth disease virus
  • various models have been proposed for to account for the“cleavage” activity: proteolysis by a host cell proteinase, autoproteolysis or a translational effect (Donnelly et al (2001) J. Gen. Virol. 82:1027-1041 incorporated herein by reference).
  • the exact mechanism of such“cleavage” is not important for the purposes of the present invention, as long as the cleavage site, when positioned between nucleic acid sequences which encode proteins, causes the proteins to be expressed as separate entities.
  • the self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus.
  • a variant can be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), preferably a variant is expressed in terms of sequence identity.
  • Sequence comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate sequence identity between two or more sequences.
  • the FOXP3 polypeptide expressed from the present vector may be positioned at the N-terminal of a self-cleaving peptide, for example a 2A self-cleaving peptide.
  • a FOXP3-2A polypeptide may comprise a sequence shown as SEQ ID NO: 5 or 6; or a variant of SEQ ID NO: 5 or 6 which is at least 80 % identical thereto.
  • the variant may be at least 85, 90, 95, 98 or 99% identical to SEQ ID NO: 5 or 6.
  • the polynucleotide encoding FOXP3 is introduced into the isolated Tregs by viral transduction.
  • Viral delivery systems include but are not limited to adenovirus vector, an adeno-associated viral (AAV) vector, a herpes viral vector, retroviral vector, lentiviral vector, baculoviral vector.
  • AAV adeno-associated viral
  • the polynucleotide encoding FOXP3 is introduced into the isolated Tregs by retroviral transduction.
  • Retroviruses are RNA viruses with a life cycle different to that of lytic viruses.
  • a retrovirus is an infectious entity that replicates through a DNA intermediate. When a retrovirus infects a cell, its genome is converted to a DNA form by a reverse transcriptase enzyme. The DNA copy serves as a template for the production of new RNA genomes and virally encoded proteins necessary for the assembly of infectious viral particles.
  • retroviruses for example murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV) and all other retroviridiae including lentiviruses.
  • retroviruses 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763, incorporated herein by reference).
  • Lentiviruses also belong to the retrovirus family, but they can infect both dividing and non dividing cells (Lewis et al. 1992 EMBO J. 3053-3058, incorporated herein by reference).
  • viral particles may be packaged with amphotropic envelopes or gibbon ape leukemia virus envelopes.
  • the method according to the invention comprises:
  • the expression“isolating the Treg from a cell population” means to separate out the Treg from a heterogeneous mixture of multiple different types of cells. Suitable the cell population is from a sample from a human subject.
  • the Treg is isolated as a population of Tregs.
  • the population of Tregs comprises at least 70 % Tregs, such as 75 %, 85 %, 90 % or 95 % Tregs.
  • the cell population comprises or consists of peripheral blood mononuclear cells (PBMCs).
  • PBMC peripheral blood mononuclear cells
  • a PBMC is any blood cell with a round nucleus found within the circulating pool of blood, rather than sequestered in the bone marrow, liver, spleen or lymphatic system.
  • PBMCs consist of monocytes and lymphocytes (T cells, B cells and NK cells).
  • T cells, B cells and NK cells monocytes and lymphocytes
  • Techniques for isolation of PBMCs from whole blood are known in the art.
  • PBMCs can be separated from a blood sample by addition of a density gradient medium, such as Ficoll (GE Healthcare), followed by centrifugation.
  • the different types of cells in the blood separate out into different layers, including a layer containing the PBMCs.
  • isolating the Treg comprises isolating CD4 + T cells. In some embodiments, isolating the Treg comprises isolating CD4 + T cells and subsequently isolating the Treg from the CD4 + T cells.
  • CD4 (cluster of differentiation 4) is a co-receptor of the T cell receptor expressed by various types of T cells. Isolation of CD4 + cells separates T cells, including Tregs, from the initial cell population. The Tregs may then be isolated from this T cell-enriched population.
  • isolating the population of Tregs comprises using immuno-magnetic beads.
  • immuno-magnetic beads Various companies (e.g. Miltenyi Biotec, Stem Cell Technologies, ThermoFisher Scientific) offer kits comprising immuno-magnetic beads for isolation of specific types of T cells (see, for example, Fallarino et al. (2003) Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4: 1206-1212, incorporated herein by reference). These isolation kits make use of antibodies widely available in the art to T cell surface proteins such as CD8, CD25, CD49b and others.
  • CD4 + cells may be first negatively selected by incubating the cell population with biotin-conjugated antibodies to markers of non-CD4 + cells (e.g. CD8) and removing these cells using anti-biotin magnetic beads. Then, Tregs may be positively selected by incubation with anti-CD25-labelled beads.
  • biotin-conjugated antibodies to markers of non-CD4 + cells e.g. CD8
  • Tregs may be positively selected by incubation with anti-CD25-labelled beads.
  • isolating the population of Tregs comprises fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • the Tregs are sorted according to their CD4 + CD25 hl CD127 phenotype.
  • Natural Tregs may be sorted from induced Tregs on the basis of expression of Helios protein or Neuropilin 1.
  • the natural Tregs may be sorted according to their CD4 + CD25 + FOXP3 + Helios + Neuropilin1 + phenotype.
  • FACS is a form of flow cytometry which is well-known in the art. During FACS, cells are suspended in fluid and streamed through a detection system that analyses various characteristics. Cells can be sorted according to their characteristics using this method. In particular, in FACS, molecules are marked using fluorescent antibodies and cells sorted according to their degree of fluorescence, which indicates level of expression of the particular molecule (see Adan et al. Flow cytometry: basic principles and applications Crit. Rev. Biotechnol. 2017 Mar;37(2): 163-176, incorporated herein by reference).
  • the invention also provides an engineered Treg, such as engineered Treg produced by the method of the invention.
  • engineered Treg means a Treg that has been manipulated by human intervention such that its gene expression has been altered.
  • the invention also provides an engineered Treg having higher FOXP3 expression than a non-engineered Treg.
  • the invention also provides a Treg having higher FOXP3 expression than a corresponding, non-engineered Treg.
  • Higher FOXP3 expression means levels of FOXP3 mRNA or protein in the engineered Treg are higher than they were before the Treg was manipulated by human intervention to alter its gene expression.
  • The“higher FOXP3 expression” may be defined and determined as described herein.
  • the level of CD25 mRNA and/or protein in a Treg modified by a method of the invention may be increased to at least 1.5-fold greater than the level in a corresponding Treg which has not been modified by a method of the invention (or population of such Tregs).
  • the level of CD25 mRNA and/or protein in a Treg modified by a method of the invention may be increased to at least 2-fold greater than the level in a corresponding Treg which has not been modified by a method of the invention (or population of such Tregs).
  • the level of CD25 mRNA and/or protein in a Treg modified by a method of the invention may be increased to at least 5-fold greater than the level in a corresponding Treg which has not been modified by a method of the invention (or population of such Tregs).
  • the level of CTLA-4 mRNA and/or protein in a Treg modified by a method of the invention may be increased to at least 1.5-fold greater than the level in a corresponding Treg which has not been modified by a method of the invention (or population of such Tregs).
  • the level of CTLA-4 mRNA and/or protein in a Treg modified by a method of the invention may be increased to at least 2-fold greater than the level in a corresponding Treg which has not been modified by a method of the invention (or population of such Tregs).
  • the level of CTLA-4 mRNA and/or protein in a Treg modified by a method of the invention may be increased to at least 5-fold greater than the level in a corresponding Treg which has not been modified by a method of the invention (or population of such Tregs).
  • the engineered Treg comprises an exogenous polynucleotide encoding a FOXP3 polypeptide.
  • exogenous polynucleotide is a polynucleotide that originates outside the Treg.
  • the exogenous polynucleotide may be introduced into the Treg as part of an expression vector. Accordingly, the exogenous polynucleotide may be contiguous with expression vector elements, such as a promoter.
  • the FOXP3 polypeptide comprises an amino acid sequence which is at least 80 % identical to SEQ ID NO: 3 or 4 or a functional fragment thereof.
  • the FOXP3 polypeptide comprises an amino acid sequence which is at least 85, 90, 95, 98 or 99% identical to SEQ ID NO: 3 or 4 or a functional fragment thereof.
  • the FOXP3 polypeptide comprises SEQ ID NO: 3 or 4 or a functional fragment thereof.
  • the exogenous polynucleotide encoding FOXP3 comprises a polynucleotide sequence which is at least 80 % identical to SEQ ID NO: 1 or 2. In some embodiments of the invention, the polynucleotide encoding FOXP3 is identical to SEQ ID NO: 1 or 2.
  • the exogenous polynucleotide encoding FOXP3 is a contiguous portion of a vector.
  • the vector also encodes a T cell receptor (TCR).
  • the vector comprises a polynucleotide sequence which is at least 80 % identical to SEQ ID NO: 5. In some embodiments of the invention, the vector comprises a polynucleotide sequence identical to SEQ ID NO: 5.
  • the invention also provides a pharmaceutical composition comprising an engineered Treg of the invention.
  • Such pharmaceutical composition may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • a pharmaceutically acceptable carrier diluent, excipient or adjuvant.
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s) and other carrier agents.
  • compositions typically should be sterile and stable under the conditions of manufacture and storage.
  • Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • Sterile injectable formulations may be prepared using a non-toxic parenterally acceptable diluent or solvent.
  • a pharmaceutical composition of the present invention may include pharmaceutically acceptable dispersing agents, wetting agents, suspending agents, isotonic agents, coatings, antibacterial and antifungal agents, carriers, excipients, salts, or stabilizers which are non toxic to the subjects at the dosages and concentrations employed.
  • such a composition can further comprise a pharmaceutically acceptable carrier or excipient for use in the treatment of disease that that is compatible with a given method and/or site of administration, for instance for parenteral (e.g. sub-cutaneous, intradermal, or intravenous injection) or intrathecal administration.
  • a pharmaceutically acceptable carrier or excipient for use in the treatment of disease that is compatible with a given method and/or site of administration, for instance for parenteral (e.g. sub-cutaneous, intradermal, or intravenous injection) or intrathecal administration.
  • composition may be produced using current good manufacturing practices (cGMP).
  • the pharmaceutical composition comprising an engineered Treg may comprise an organic solvent, such as but not limited to, methyl acetate, dimethyl sulfoxide (DMSO), N,N- dimethylformamide (DMF), dimethoxyethane (DME), and dimethylacetamide, including mixtures or combinations thereof.
  • an organic solvent such as but not limited to, methyl acetate, dimethyl sulfoxide (DMSO), N,N- dimethylformamide (DMF), dimethoxyethane (DME), and dimethylacetamide, including mixtures or combinations thereof.
  • the pharmaceutical composition is endotoxin free.
  • the invention also provides an engineered Treg of the invention, or a pharmaceutical composition of the invention, for use in prevention and/or treatment of a disease.
  • the invention also provides use of an engineered Treg of the invention in the manufacture of a medicament for prevention and/or treatment of a disease.
  • the invention also provides a method of prevention and/or treatment of a disease comprising administering to a subject an engineered Treg or a composition of the invention.
  • the method of prevention and/or treatment of a disease comprises administration of a pharmaceutical composition of the present invention to a subject.
  • treat/treatment/treating refers to administering an engineered Treg or pharmaceutical composition of the invention to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
  • PreventionYpreventing refers to delaying or preventing the onset of the symptoms of the disease. Prevention may be absolute (such that no disease occurs) or may be effective only in some individuals or for a limited amount of time.
  • the subject of the method of the invention is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit or guinea pig.
  • the subject is a human.
  • an engineered Treg or pharmaceutical composition can be administered intravenously, intrathecally, by oral and parenteral routes, intranasally, intraperitoneally, subcutaneously, transcutaneously or intramuscularly.
  • the engineered Treg or pharmaceutical composition of the invention is administered intravenously.
  • the engineered Treg or pharmaceutical composition of the invention is administered intrathecally.
  • a physician will determine the dosage that is most suitable for an individual subject, and the dosage will vary with the age, weight and response of the particular subject.
  • the dosage is such that it is sufficient to reduce and/or prevent disease symptoms.
  • route of delivery e.g. oral vs. intravenous vs. subcutaneous etc.
  • focussed delivery may be preferred.
  • Other factors to be considered when optimizing routes and/or dosing schedule for a given therapeutic regimen may include, for example, the disease being treated (e.g. type or stage etc.), the clinical condition of a subject (e.g. age, overall health etc.), the presence or absence of combination therapy, and other factors known to medical practitioners.
  • the dosage is such that it is sufficient to stabilise or improve symptoms of the disease.
  • the present invention also provides a method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition comprising a cell e.g. a T cell according to the invention to a subject.
  • the method of prevention and/or treatment of a disease of may comprise:
  • Tregs may be isolated from a patent by taking a blood sample and isolating Tregs from it using techniques known in the art, such as those described in this specification under the heading“Isolating Tregs”.
  • a polynucleotide encoding a FOXP3 polypeptide may be introduced into Tregs using techniques known in the art, such as those described in this specification under the heading “Viral transduction”.
  • the engineered Tregs may be expanded in vitro before administration to the subject.
  • Tregs may be expanded in vitro by culturing them in TexMACX® media.
  • the disease to be treated and/or prevented by the methods and uses of the present invention may be any disease which is associated with a pathological immune response.
  • the disease may be, for example, a cancer, infectious disease or autoimmune disease.
  • the disease is an autoimmune disease.
  • the disease may have central nervous system (CNS) involvement of systemic autoimmune and inflammatory disease such as Behcet disease, sarcoidosis, systemic lupus erythematosus, juvenile idiopathic arthritis, scleroderma, and Sjogren syndrome.
  • CNS central nervous system
  • the disease may be any disease wherein MBP is an antigen e.g. where MBP is a self antigen.
  • the disease may be an autoimmune and inflammatory central nervous system disease (e.g. chronic neurodegenerative conditions).
  • central nervous system disease e.g. chronic neurodegenerative conditions.
  • the disease may be a chronic neurodegenerative condition such as multiple sclerosis (MS), Alzheimer’s disease, Parkinson’s disease, neurotropic viral infections, stroke, paraneoplastic disorders and traumatic brain injury.
  • MS multiple sclerosis
  • Alzheimer’s disease Parkinson’s disease
  • neurotropic viral infections stroke, paraneoplastic disorders and traumatic brain injury.
  • the disease is multiple sclerosis.
  • the disease is chronic progressive multiple sclerosis.
  • the disease is relapsing/remitting multiple sclerosis.
  • the disease is present in an HLA-DRB1*0401 positive subject.
  • the disease is multiple sclerosis and the subject is HLA-DRB1*0401 positive.
  • the disease is chronic progressive multiple sclerosis and the subject is HLA-DRB1*0401 positive.
  • the disease is relapsing/remitting multiple sclerosis and the subject is HLA-DRB1*0401 positive.
  • MS Multiple Sclerosis
  • MS is the most common neurological disorder among young adults in Europe and in the USA.
  • MS is characterised as a demyelinating disease and is a chronic degenerative disease of the central nervous system in which gradual destruction of myelin occurs in patches throughout the brain and/ or spinal cord, interfering with neural connectivity and causing muscular weakness, loss of coordination and speech and visual disturbances.
  • CIS clinically isolated syndrome
  • RRMS relapsing-remitting MS
  • PPMS primary progressive MS
  • SPMS secondary progressive MS
  • DMTs disease-modifying treatments
  • Neither DMTs nor stem cell transplantation can mediate antigen-specific suppression of the immunopathology of MS.
  • administration of one dose of the engineered Treg of the present invention may provide lasting suppression of MS immunopathology in the absence of systemic side effects. This will have a significant impact on the progression of the disease in people with MS.
  • the engineered Treg or pharmaceutical composition of the present invention may reduce or ameliorate one or more of the symptoms of MS, which include reduced or loss of vision, stumbling and uneven gait, slurred speech, urinary frequency and incontinence, mood changes and depression, muscle spasms and paralysis.
  • the present invention further provides a method for inducing tolerance to a transplant; treating and/or preventing cellular and/or humoral transplant rejection; treating and/or preventing graft-versus-host disease (GvHD), which comprises the step of administering an engineered Treg or a pharmaceutical composition of the invention to a subject.
  • GvHD graft-versus-host disease
  • inducing tolerance to a transplant refers to inducing tolerance to a transplanted organ in a recipient.
  • inducing tolerance to a transplant means to reduce the level of a recipient’s immune response to a donor transplant organ.
  • Inducing tolerance to a transplanted organ may reduce the amount of immunosuppressive drugs that a transplant recipient requires, or may enable the discontinuation of immunosuppressive drugs.
  • the subject is a transplant recipient undergoing immunosuppression therapy.
  • the transplant may be selected from a liver, kidney, heart, lung, pancreas, intestine, stomach, bone marrow, vascularized composite tissue graft, and skin transplant.
  • Embodiments of the invention may be combined.
  • CD4+ T cells were isolated using a CD4+ Positive selection kit. Cells were subsequently stained with flow cytometry antibodies CD4, CD25 and CD127 before FACS sorting using the BD ARIA. CD4+CD25hiCD127- Treg and CD4+CD25-CD127+ Tconv were collected in polypropylene tubes. Purity of cell sorting was determined by addition of FOXP3 PE antibody. Purity of CD4+CD25+CD127-FOXP3+ cells was routinely >70 %.
  • Treg and Tconv were activated separately for 48 hours by culturing 1 :1 with anti-CD3 and anti-CD28 beads.
  • cells were counted and resuspended in complete RPMI (Tconv) or Texmacs media (Treg) at 1x10 6 /ml_.
  • Non-tissue culture-treated 24-well plates were pre-prepared by coating with retronectin then subsequently blocked with 2 % bovine serum albumin in PBS and washed x2 with PBS.
  • Final concentration of IL-2 was 300 m/ml for Tconv and 1000 m/ml for Treg.
  • Cells were incubated overnight at 37 °C before removing supernatant and supplementing with fresh complete media and IL-2. Media was changed on alternate days.
  • Tconv cells were grown in RPMI-1640 (Gibco) supplemented with 10 % heat inactivated foetal bovine serum; 100 Units/mL penicillin; 100 pg/mL streptomycin; 2 mM L-glutamine. Regulatory T cells were cultured in Texmacs media (Miltenyi) supplemented with 100 Units/mL penicillin; 100 pg/mL streptomycin.
  • Example 1C Proliferation and IL-2 production of stimulated Tconv cells in the presence of FOXP3-transduced natural Tregs
  • Transduced responder T cells were stained with CFSE cell trace dye in warmed PBS at 37 °C for 3 minutes before addition of equal volumes of warm FBS and a further 3 minute incubation. Cells were washed in 5x volume of complete RPMI media before counting and resuspension at 1x10 6 transduced cells/ml. Regulatory T cells were removed from culture, washed and re-suspended at 1x10 6 transduced cells/ml in complete RPMI. Cells were plated 1 Treg : 0.1 CHO cells : varying ratios of Tconv for 4 days.
  • FIG. 1 shows the proliferation of TCR transduced Tconv cells with and without peptide (blue bars) and the proliferation of the same cells in the presence of Mock Treg (white bars), TCR transduced Treg or TCR+FOXP3 transduced Treg.
  • Figure 2 shows the IL-2 production of TCR transduced Tconv cells with and without peptide (blue bars) and the proliferation of the same cells in the presence of Mock Treg (white bars), TCR transduced Treg or TCR+FOXP3 transduced Treg.
  • Example 2 T cells from a different donor
  • Example 1 The experiment described in Example 1 was repeated using T cells from a different donor.
  • Figure 3 shows percentage proliferation of TCR transduced T cells.
  • Figure 4 shows the concentration of IL-2 in supernatants collected from co-culture experiments.
  • Treg markers (FOXP3, CD25 and CTLA-4) at day 7-10.
  • Figure 5 shows the mean fluorescence intensity (MFI) of each marker. Dots are representative of individual experiments. 1-way ANOVA was used for statistical analysis p ⁇ 0.05 *, p ⁇ 0.005 **.
  • FIG. 6 represents the same data differently.
  • Each line represents a single experiment showing MFI of markers on the same Treg transduced with TCR or TCR+FOXP3.
  • CD80 + CD86 + DR4 + CHO cells were loaded with peptide and irradiated as described in Example 1C before being re-suspended at 0.1x10 6 cells/ml.
  • Transduced responder T cells were stained with CFSE cell trace dye in warmed PBS at 37 °C for 3 minutes before addition of equal volumes of warm FBS and a further 3 minute incubation.
  • Tregs were washed in 5x volume of complete media before being counted and resuspended at 1x10 6 transduced cells/ml.
  • the transduction efficiency of Tconv and Treg were determined by flow cytometry. Tregs were removed from culture, washed and resuspended at 1x10 6 transduced cells/ml in complete RPMI. Cells were plated 1 Treg : 0.1 CHO cells : varying parts Tconv. Proliferation was determined by analysing dilution of carboxyfluorescein succinimidyl ester (CFSE)-stained Tconv.
  • CFSE carboxyfluorescein succinimidyl ester
  • Thy1 .1 +CD4+CD25+ or CD45.1 +CD4+CD25+ Treg were isolated from lymph nodes and splenocytes of HLA-DRB * 0401 transgenic mice by bead sort.
  • CD45.1 + Treg were transduced with TCR and Thy1.1 + Treg were transduced with TCR+murine FOXP3.
  • TCR or TCR+FOXP3 transduced cells were injected in a 1 : 1 ratio into FILA-DRB * 0401 transgenic hosts conditioned with 4Gy irradiation.
  • FACS plots show the ratio of CD45.1 :Thy1 .1 of injected cells and their respective FOXP3 expression.
  • Thy1.1+CD4+CD25+ Treg were isolated from lymph nodes and splenocytes of HLA- DRB*0401 transgenic mice by bead sort. Treg were transduced TCR, TCR+murine FOXP3 or cultured with virus-free supernatant (mock). 1 day after transduction TCR or TCR+FOXP3 transduced cells were injected into HLA-DRB*0401 transgenic hosts conditioned with 4Gy irradiation. 7 weeks later flow cytometry was used to determine the engraftment of transduced Treg Figure 9, A shows the transduction efficiency determined through expression of human variable 2.1 and murine Foxp3 on d1 post-transduction.
  • Figure 9, B shows splenocytes from mice that received Treg transduced with TCR or TCR+FOXP3 stained with Thy1.1 to identify transferred cells (top panel) and FOXP3 and TCR (bottom panel).
  • Figure 9, C shows cumulative data showing fold change in transduction efficiency (left panel) and fold change in absolute number of transduced cells (right panel) relative to day of injection for Treg transduced with TCR or TCR+FOXP3.
  • Figure 9, D shows a representative expression of FOXP3 within transduced cells 7 weeks after transfer. Graphs show cumulative of percentage FOXP3+ cells within the transduced population at week 7 (left) and the fold change in FOXP3+ cells relative to the day of injection.
  • Example 6B - Treg expressing exogenous FOXP3 retain Treg functionality after 7 weeks in vivo whilst Tregs not expressing exogenous FOXP3 acquire the ability to produce effector cytokines
  • Treg expressing exogenous FOXP3 retain Treg functionality after 7 weeks in vivo as demonstrated by lack of effector cytokine production, whilst Tregs not expressing exogenous FOXP3 acquire the ability to produce effector cytokines (Figure 10).

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Abstract

La présente invention concerne une méthode pour améliorer la capacité de lymphocytes T régulateurs (Treg) à supprimer des réponses immunitaires comprenant l'augmentation de l'expression de FOXP3 dans une population de Treg.
EP19719600.9A 2018-04-18 2019-04-17 Méthode permettant d'améliorer les propriétés suppressives de lymphocytes treg Pending EP3781673A1 (fr)

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